Showing papers on "Sperm plasma membrane published in 2002"

Control of Membrane Fusion During Spermiogenesis and the Acrosome Reaction

Abstract: Membrane fusion is important to reproduction because it occurs in several steps during the process of fertilization. Many events of intracellular trafficking occur during both spermiogenesis and oogenesis. The acrosome reaction, a key feature during mammalian fertilization, is a secretory event involving the specific fusion of the outer acrosomal membrane and the sperm plasma membrane overlaying the principal piece of the acrosome. Once the sperm has crossed the zona pellucida, the gametes fuse, but in the case of the sperm this process takes place through a specific membrane domain in the head, the equatorial segment. The cortical reaction, a process that prevents polyspermy, involves the exocytosis of the cortical granules to the extracellular milieu. In lower vertebrates, the formation of the zygotic nucleus involves the fusion (syngamia) of the male pronucleus with the female pronucleus. Other undiscovered membrane trafficking processes may also be relevant for the formation of the zygotic centrosome or other zygotic structures. In this review, we focus on the recent discovery of molecular machinery components involved in intracellular trafficking during mammalian spermiogenesis, notably related to acrosome biogenesis. We also extend our discussion to the molecular mechanism of membrane fusion during the acrosome reaction. The data available so far suggest that proteins participating in the intracellular trafficking events leading to the formation of the acrosome during mammalian spermiogenesis are also involved in controlling the acrosome reaction during fertilization.

GPX5 is present in the mouse caput and cauda epididymidis lumen at three different locations.

Abstract: In mice, GPX5 is a secreted protein abundantly synthesized by the caput epididymidis. The protein is secreted as early as the initial segment of the caput and is found subsequently associated with the sperm plasma membrane in a sub-acrosomic localization. We show here that GPX5 is present in the caput and cauda epididymides lumens in three different locations: either free as a soluble protein in the caput epididymal fluid, weakly bound to caput sperm membranes, or, finally, associated to lipid-containing structures conferring to the protein a protective effect against proteolytic digestions. Within the cauda epididymidis, the amount of free GPX5 is low compared to the caput and the association with sperm membranes proved to be more solid. In both caput and cauda sperm samples, the association of GPX5 with the sperm membrane protects GPX5 from proteolytic cleavages. Protection against proteolytic digestions can be overcome by physical treatments of epididymal fluid and sperm samples such as ultrasounds or very acidic pH. These data suggest that complex phenomena and structures participate in the transfer and binding of the caput-secreted GPX5 protein to the sperm plasma membrane.

Sperm plasma-membrane-associated glutathione S-transferases as gamete recognition molecules.

Abstract: Glutathione S-transferases (GSTs) are enzymes that detoxify electrophilic compounds. Earlier studies from our laboratory showed that anti-GST antibodies interfered with the fertilising ability of spermatozoa from Capra hircus (goat) in vitro, suggesting that GSTs are localised at the cell surface. In this study, we provide evidence for the presence of GSTs of 24 kDa on the sperm plasma membrane attached by non-covalent interactions. The GST activity associated with the spermatozoal plasma membrane was significantly higher than the activity present in the plasma membranes of brain cells, hepatocytes, spleenocytes and ventriculocytes. Analysis of GST isoforms demonstrates the presence of GST Pi and Mu on the sperm plasma membranes. Both isoforms were able to bind to solubilised as well as intact zona pellucida (ZP) through their N-terminal regions but failed to bind to ZP once the oocytes were fertilised. Solubilised goat ZP separates into three components, one of which, the ZP3-like component, bound to sperm GSTs. High concentrations of anti-GST antibodies or solubilised ZP led to aggregation of sperm GSTs, resulting in the release of acrosin. In contrast, inhibition of sperm GST binding to ZP, by saturation of binding sites for sperm GSTs on the solubilised ZP using peptides designed from the N-terminii of GST Pi or Mu or blocking of binding sites for ZP on sperm GSTs with antibodies raised against the N-terminal GST peptides, inhibited essential prefertilisation changes in sperm. These data therefore demonstrate the strategic location of catalytically active defensive enzymes on the sperm surface that also act as zona-binding proteins. Therefore, sperm-surface GSTs serve as bifunctional molecules in a transcriptionally inactive cell whose requirement for cellular defense and economy of molecules that it can carry is greater than that of any somatic cell type.

Role of Sperm Surface Arylsulfatase A in Mouse Sperm-Zona Pellucida Binding

Abstract: We have previously described the zonae pellucidae (ZP) binding ability of a pig sperm surface protein, P68. Our recent results on peptide sequencing of 3 P68 tryptic peptides and molecular cloning of pig testis arylsulfatase A (AS-A) revealed the identity of P68 as AS-A. In this report, we demonstrate the presence of AS-A on the mouse sperm surface and its role in ZP binding. Using anti-AS-A antibody, we have shown by immunoblotting that AS-A was present in a Triton X-100 extract of mouse sperm. The presence of AS-A on the sperm plasma membrane was conclusively demonstrated by indirect immunofluorescence, immunogold electron microscopy, and AS-A's desulfation activity on live mouse sperm. The AS-A remained on the head surface of in vivo capacitated sperm, as revealed by positive immunofluorescent staining of oviductal/uterine sperm. Significantly, the role of mouse sperm surface AS-A on ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding on sperm pretreatment with anti-AS-A IgG/Fab. Furthermore, Alexa-430 conjugated AS-A bound to mouse ZP of unfertilized eggs but not to fertilized ones, and this level of binding increased and approached saturation with increasing Alexa-430 AS-A concentrations. Moreover, in vivo fertilization was markedly decreased when mouse sperm pretreated with anti-AS-A IgG were artificially inseminated into females. All of these results designated a new function for AS-A in mouse gamete interaction.

Osmotic characteristics of mouse spermatozoa in the presence of extenders and sugars.

Abstract: Successful cryopreservation requires cells to tolerate volume excursions experienced during permeating cryoprotectant equilibration and during cooling and warming. However, prior studies have demonstrated that mouse spermatozoa are extremely sensitive to osmotically induced volume changes. A series of three experiments were conducted 1) to test the efficacy of two commonly used extender media components, egg yolk (EY) and skim milk (SM), in broadening the osmotic tolerance limits (OTL) of ICR and B6C3F1 murine spermatozoa; 2) to determine if the extender components affected sperm plasma membrane permeability coefficients for water and cryoprotective agent (CPA) characteristics; and 3) to test the effects of permeating and nonpermeating CPA on mouse sperm morphology. In experiment 1, sperm samples were added to 150, 225, 300, 450, or 600 mOsm NaCl, EY, SM, sucrose, or choline chloride at 22°C and then returned to isosmotic conditions. In experiment 2, epididymal sperm were preequilibrated in 1 M g...

Importance of cooling rate and animal variability for boar sperm cryopreservation: insights from the cryomicroscope

Abstract: A series of experiments was set up to investigate the effect of different cooling rates on boar sperm cryosurvival using cryomicroscopy. The cooling protocols were split into two stages: (i) from +5 degrees C to -5 degrees C and (ii) from -5 degrees C to -50 degrees C. Fluorescent probes (SYBR14 and propidium iodide) were used to monitor plasma membrane integrity during the entire process. Cooling rates in the range 3 degrees C min(-1) to 12 degrees C min(-1) did not cause significant damage to the sperm plasma membrane between +5 degrees C and -5 degrees C; however, spermatozoa cooled at 24 degrees C min(-1) to -5 degrees C were slightly damaged. Motility was not particularly sensitive to variations in cooling rate. Cooling rates in the range 15 degrees C min(-1) to 60 degrees C min(-1) did not produce differences in sperm cryosurvival during freezing between -5 degrees C and -50 degrees C, or after thawing. In addition, cooling rates in the range 3 degrees C min(-1) to 80 degrees C min(-1) did not produce significant differences in sperm cryosurvival. However, slow freezing (3 degrees C min(-1)) induced a slight increase in the percentage of plasma membrane-damaged spermatozoa (propidium iodide-positive) at -50 degrees C. Inter-ejaculate and inter-boar differences in sperm cryosurvival were manifested independently of cooling rate. The sperm plasma membrane remained intact (SYBR14-positive) during cooling and freezing, but upon rewarming, the plasma membrane of a high proportion of spermatozoa was damaged (propidium iodide-positive), indicating that rewarming is a critical step of the freezing-thawing process.

Voltage-operated calcium channels in male germ cells

Abstract: The acrosome reaction is a key event in fertilization. Current models for induction of the acrosome reaction incorporate a necessary influx of Ca 2+ , which is mediated by agonist-induced gating of ion channels in the sperm plasma membrane. The difficulty of applying electrophysiological techniques to spermatozoa has severely hampered studies on the expression of functional ion channels in these cells. However, during the last few years, a combination of molecular and physiological techniques (applied to immature spermatogenic cells) has elucidated both the expression of Ca 2+ channels in male germ cells and their role in induction of the acrosome reaction. It now appears that a range of voltage-operated Ca 2+ channels, similar to those that occur in somatic cells, is expressed in spermatozoa. Male rodent germ cells express a low-voltage activated (T-type) channel that is regulated by membrane potential and provides the primary Ca 2+ influx mechanism in zona pellucida-stimulated spermatozoa. In human spermatozoa, similar channels are apparently expressed, but their function in induction of the acrosome reaction has yet to be established. A range of other, high voltage-activated channels also appear to be present in rodent and human spermatozoa, but their roles are not yet known. In this review, the structure and characteristics of voltage-operated Ca 2+ channels are outlined and the evidence for their expression and function in male germ cells is assembled and discussed.

Identification of Ras and Its Downstream Signaling Elements and Their Potential Role in Hamster Sperm Motility

Abstract: Ras, a member of the small G-protein family, regulates multiple signaling pathways in somatic cells. The objectives of the present study included the characterization and localization of Ras and the identification of its downstream effectors in hamster spermatozoa. Immunoblot analysis with a pan-Ras monoclonal antibody localized Ras to the particulate fraction of sonicated testicular and caput and cauda epididymal spermatozoa. However, Ras was present in both the particulate and soluble fractions of spermatocytes and round spermatids, suggesting that its membrane recruitment is completed during spermiogenesis. Immunoblots of plasma membrane fractions demonstrated that hamster spermatozoa express both N-Ras and K-Ras. Indirect immunofluorescence with pan-Ras antibody localized Ras to the flagellum. Immunoblot analysis of sperm plasma membrane fractions demonstrated the presence of phosphatidylinositol 3kinase (PI3-kinase) and protein kinase C z (PKCz), the downstream targets of Ras, and coimmunoprecipitation analysis demonstrated their interaction with Ras. Inhibitors of PI3-kinase (wortmannin and 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) and PKCz (staurosporine) inhibited the hyperactivation of sperm motility during capacitation in a dose-dependent manner, indicating that both PI3-kinase and PKCz are associated with development of this motility pattern. The interaction of Ras with both PI3-kinase and PKCz suggests that Ras may regulate several signaling pathways in spermatozoa. gamete biology, signal transduction, sperm maturation, sperm motility and transport, spermatogenesis

An anti-actin monoclonal antibody inhibits the zona pellucida-induced acrosome reaction and hyperactivated motility of human sperm

Abstract: We report an inhibitory effect of an anti-actin monoclonal antibody (mAb) on the human zona pellucida (ZP)-induced acrosome reaction (AR). Motile sperm were incubated with native human ZP for 2 h in medium containing either the anti-actin mAb, an irrelevant control mAb or cytochalasins B or D (40 micromol/l). Sperm bound to the ZP were recovered and the AR was determined by fluorescein-labelled Pisum Sativum agglutinin. Anti-mouse immunoglobulin G (mIgG) Dynabeads, immunofluorescence and immunogold were used to detect the location of the anti-actin mAb in sperm. The anti-actin mAb significantly inhibited the ZP-induced AR (equivalent to cytochalasins), the ionophore A23187-induced AR and hyperactivation of sperm in medium. After incubation with anti-actin mAb, anti-mIgG beads bound to the head of >50% of sperm recovered after binding to the ZP and 10% of sperm remaining in the medium. The proportion of sperm that bound anti-mIgG beads after recovery from binding to the ZP in the presence of the anti-actin mAb was significantly correlated with the ZP-induced AR in the absence of the antibody. Immunofluorescence and immunogold demonstrated entry of the anti-actin mAb into sperm. This study suggests that the sperm plasma membrane becomes permeable to the anti-actin mAb during capacitation and initiation of the AR.

Bull semen evaluation post-thaw and relation of semen characteristics to bulls fertility

Abstract: Evaluation of the quality of cryopreserved bull spermatozoa is reviewed. The methods for the assessment of sperm quality have markedly improved over the last decades - starting with the assessment of morphological shapes and subjective motility analysis towards the more sophisticated analysis of the molecular changes in chromatin, membranes and catabolic activities of the sperm cell itself. Function of sperm plasma membrane under the hypo- osmotic conditions, distribution and concentration of ions, or function of different organelles seem to correlate with the degree of the viability of spermatozoa after freezing and thawing procedures. Many in vitro techniques that stimulate sperm function through female-derived factors, such as zona and oocyte, in zona pellucida binding assay, in vitro fertilization (IVF) and production of embryos, were employed to predict the outcome of artificial insemination in the field. Still, majority of methods used for semen analysis today are both - tedious and expensive, and, in many cases confined to human bias. In order to increase the predictive power of assessment, simultaneous analysis of multiple sperm attributes, or outcomes of several laboratory assessments must be combined to look for the overall effect of several independent sperm parameters.

Structural Differentiation of Spermatozoa During Post-Testicular Maturation

Abstract: The male gamete exits from the testis as a highly polarized but functionally immature spermatozoon that requires further differentiation in the epididymis to become progressively motile and to acquire fertilizing capacity. This crucial developmental process requires sperm interaction with a progressively changing luminal environment regulated by region-specific secretory and absorptive activities of the epididymal epithelium (Hinton and Palladino, 1995; Hinton et al., 1996). Early studies demonstrated striking differences in the electrophoretic mobility of whole caput and cauda epididymal spermatozoa (Bedford, 1963), suggesting that a maturation-dependent increase in anionic residues on the sperm surface resulted from this interaction. This idea was confirmed ultrastructurally by cytochemical studies demonstrating altered binding of charged colloidal iron particles and lectins to specific surface domains of the spermatozoon as they transit the epididymis (Yanagimachi et al., 1972; Nicolson et al., 1977). Now it is recognized that an extensive biochemical remodeling of the protein, glycoprotein and lipid composition of the sperm plasma membrane is an important component of post-testicular sperm maturation (Bartles, 1995; Jones 1998; Ladha, 1998).

Lipid diffusion in the plasma membrane of mouse spermatozoa: changes during epididymal maturation, effects of pH, osmotic pressure, and knockout of the c-ros gene.

Abstract: It is well known that the plasma membranes of mammalian spermatozoa undergo extensive remodeling during maturation in the epididymal duct. In this investigation, we have used fluorescence recovery after photobleaching (FRAP) techniques to: 1) measure rates of lipid diffusion in the plasma membrane of mouse spermatozoa at different stages of maturation; 2) examine the effects of varying external conditions found in the epididymal duct (pH, temperature, and osmotic pressure) on lipid diffusion in mature sperm; and 3) investigate the effects of the c-ros null mutation that causes tail angulation in cauda spermatozoa after ejaculation as a result of cell swelling due to altered membrane function. Our results show that lipid diffusion (as measured using reporter probes 5-(N-octadecanoyl)aminofluorescein [ODAF] and 2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phos-phocholine [NBD-C6-PC]) is several times faster across the membrane on the sperm head than on the tail and that it increases significantly during passage from caput to cauda. Temperature variations between 20°C and 37°C have a substantial effect on diffusion coefficients, with the sperm head being more responsive than the tail. Changes in external pH (6.5–8.5) or osmotic pressure (202–389 mOsm/kg), however, have little relative effect on lipid diffusion on any region of the sperm. The rate of diffusion of 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3βol (NBD-cholesterol) is 10-fold higher across the head plasma membrane than across the tail and does not change significantly during epididymal maturation. Similarly, lipid diffusion in hairpin-shaped cauda sperm from c-ros (-/-) males is not significantly different from (+/+) controls. These results suggest that temperature and compositional changes are 2 of the important factors that regulate the dynamics of lipid molecules in the mouse sperm plasma membrane.

Importance of Glycosylation and Disulfide Bonds in Hyaluronidase Activity of Macaque Sperm Surface PH-20

Abstract: PH-20 is a glycoprotein located on the surface of the sperm plasma membrane and on the inner acrosomal membrane. The best understood function of sperm surface PH-20 is its hyaluronidase activity, which results in hydrolysis of the hyaluronic acid-rich cumulus matrix during sperm penetration of this extracellular oocyte investment. In this study, we investigated whether alterations in the secondary and tertiary structures of sperm surface PH-20 would affect its enzyme activity. Proteins were isolated from the sperm plasma membrane by treatment of living cells with phosphatidylinositol-specific phospholipase C (PI-PLC). PH-20 was purified from the PI-PLC released proteins by immunoaffinity chromatography. Two-dimensional electrophoresis of purified PH-20 revealed 6 isoforms with isoelectric points ranging from 5.1 to 6.0. Removal of the N-linked glycans from PH-20 with N-glycosidase F shifted the molecular weight from 64 kd to approximately 54 kd, its deduced molecular weight based on sequence analysis, suggesting that most if not all, of the potential N-glycosylation sites are linked to oligosaccharides. The lectins Con A and PSA recognized purified sperm surface PH-20 after Western blotting, suggesting that mannose is a major sugar within or at the terminal end of the linked glycan. The lectins UEA and LPA did not recognize PH-20 Western blot, suggesting that fucose and sialic acid are not terminal sugars of sperm surface PH-20. Deglycosylation of sperm surface PH-20 resulted in a complete loss of its hyaluronidase activity. The reduction of disulfide bonds with beta-mercaptoethanol or dithiothreitol also resulted in loss of enzyme activity. We conclude that the hyaluronidase activity of sperm surface PH-20 is dependent on structural features established by sulfhydryl linkages, as well as glycosylation.

The role of Na,K-ATPase in human sperm motility

Abstract: A fourth Na,K-ATPase alpha isoform, which was found to be abundant in testes, was proved to be a catalytical subunit of the enzyme. Recently, it has been shown that the alpha 4 isoform along with alpha 1 is expressed in the midpiece of the flagellum of mature rat sperm and the inhibition of alpha 4 with ouabain led to sperm immotility. In this study, sperm from 135 males with normal semen profile and 50 males with oligoasthenospermia were treated with 10-5 and 10-2 M ouabain solutions to inhibit alpha 4, and alpha 4 plus alpha 1 isoforms, respectively. In males with normal semen profile, sperm motility has been demonstrated to decrease with time to almost the same level with both ouabain solutions. In oligoasthenospermic males motility was also found almost completely lost. These observations showed us that the alpha 4 isoform may be held responsible for human sperm motility. When sperm plasma membrane Na,K-ATPase activity was assayed for both normal and oligoasthenospermic males, a significant decrease in enzyme activity of males with oligoasthenospermia was observed (p < 0.05). In our recent study, sperm motility was found decreased by treatment with peroxynitrite (ONOO-). To investigate the effect of ONOO- on sperm Na,K-ATPase activity, sperm plasma membranes were treated with 100 microM ONOO- and plasma membrane Na,K-ATPase activity was observed to be significantly decreased (p < 0.05). Although total sulfhydryl (SH) content of sperm plasma membrane was also found significantly lower, no correlation was found between Na,K-ATPase activity and SH content.

Case studies in antelope aggression control using a GnRH agonist

Abstract: Maintaining surplus captive male antelope in bachelor groups can result in aggression in some species, leading to injury or death. Suppressing endogenous testosterone using gonadotropin-releasing hormone (GnRH) analogs has been used in primates to control aggressive behavior, but little information is available on the use of GnRH analogs in nondomestic ruminant species. The aim of this study was to investigate the effect of a slow-release GnRH agonist (deslorelin) on circulating hormone concentrations, semen and sperm characteristics and behavior in male gerenuk, dorcas gazelle, and scimitar horned oryx. Body weight, testicular volume, circulating hormone concentrations, ejaculate traits, and behavior were recorded before and during deslorelin treatment. A GnRH challenge (with serial blood sampling) was administered to gerenuk and dorcas gazelles before and during GnRH analog treatment. Quantitative behavioral data were collected for gerenuk and dorcas gazelles for 30 min three times a week, starting 1 month before deslorelin treatment, and the mean incidence of combined aggressive behaviors (supplanting, foreleg kicking, sparring, marking, and mounting) was compared before and during deslorelin treatment. No statistical difference (P>0.05) in body weight, semen volume, sperm concentration, percent sperm motility, percent sperm plasma membrane integrity, or percent normal sperm morphology was found before or during deslorelin treatment. The characteristic rise in luteinizing hormone (LH), occurring ∼10 min following administration of a GnRH challenge in untreated males, was not evident during deslorelin treatment, although tonic LH concentrations were maintained. No differences (P>0.05) in the mean incidence of any aggressive behavioral traits in gerenuk or dorcas gazelle were detected before and during deslorelin. The absence of a GnRH-induced increase in serum LH in treated males indicated that deslorelin suppressed pituitary responsiveness to endogenous GnRH, but that the continued tonic production of LH was sufficient to maintain testosterone production, aggressive behavior, and subsequent semen production. Zoo Biol 21:435–448, 2002. © 2002 Wiley-Liss, Inc.

Progesterone treatment of boar spermatozoa improves male pronuclear formation after intracytoplasmic sperm injection into porcine oocytes.

Abstract: Boar spermatozoa were prepared for intracytoplasmic sperm injection (ICSI) by two different treatments to facilitate sperm chromatin decondensation and improve fertilisation rates after ICSI in pigs: spermatozoa were either frozen and thawed without cryoprotectants, or treated with progesterone. Morphological changes of the sperm heads after the treatments were examined and then the activation of oocytes and the transformation of the sperm nucleus following ICSI were assessed. After freezing and thawing, the plasma membrane and acrosomal contents over the apical region of sperm head were lost in all the spermatozoa. Following treatment with 1 mg/ml progesterone, the acrosome reaction was induced in 61% of spermatozoa. After injection of three types of spermatozoa, non-treated spermatozoa and progesterone-treated (i.e. acrosome-reacted) spermatozoa induced oocyte activation, but frozen-thawed spermatozoa induced oocyte activation at a significantly lower rate. Sixty-two per cent of sperm heads remained orcein-negative for 6 h, however, resulting in delayed sperm chromatin decondensation and low male pronuclear formation in the oocytes injected with a non-treated spermatazoon. Since the treatments of freezing and thawing and progesterone for spermatozoa accelerated the initial change in sperm chromatin and the latter treatment induced oocyte activation earlier, it is considered that the delay in oocyte activation and decondensation of sperm chromatin after injection of non-treated spermatozoa is caused by the existence of the sperm plasma membrane. These results show that progesterone treatment efficiently induces the acrosome reaction in boar spermatozoa without destroying their potency for oocyte activation, and the induction of the acrosome reaction results in the promotion of male pronuclear formation after ICSI.

Plasma Membrane Composition and Organisation During Maturation of Spermatozoa in the Epididymis

Abstract: In keeping with all somatic cells, mammalian spermatozoa are surrounded by a limiting plasma membrane (PM) containing lipids, proteins, glycolipids and glycoproteins arranged in a classic bilayer structure. Spermatozoa are also highly polarised cells and their PM is compartmentalised into different regions (acrosome, equatorial segment, postacrosome, midpiece and principal piece) that reflect compositional and functional heterogeneity. Unlike somatic cells, however, spermatozoa are exposed to widely different environments during their lifetime, ranging from the unique milieu within the seminiferous tubules to that in the epididymis, seminal plasma, uterus, oviduct, cumulous oophorus, and eventually the perivitilline space. The PM has to be sufficiently stable physiologically to withstand these varying conditions, while at the same time sensitive enough to detect specific signals from different fluids and to react accordingly. Since spermatozoa acquire their motility and fertilizing capacity during passage through the epididymal duct, much interest has centred on the nature of the PM during their transition from an infertile to a fertile state. This information is important not only from the standpoint of elucidating the biogenesis of the molecules involved in fertilization itself, but also for understanding the aetiology of infertility in man. This chapter, therefore, will focus on the PM remodelling events during passage of spermatozoa through the epididymis.

Active Oxygen in Spermatozoa During Epididymal Transit

Abstract: The spermatozoa generated by the germinal epithelium of the testis are incapable of fertilization but achieve functional competence as they mature in the epididymis. As a consequence of epididymal maturation the spermatozoa gain the ability to express coordinated flagellar beating and initiate capacitation as soon as they are released from the epididymal environment. Capacitation is in many ways an extension of the maturation process initiated in the epididymis. As a biological phenomenon, capacitation has been recognized for half a century but only recently has the biochemical basis of this process become apparent (De Lamirande et al., 1997). The need for capacitation was acknowledged when it was found that spermatozoa recovered directly from the cauda epididymidis did not have the ability to fertilize ova in vitro. Before fertilization became possible, the spermatozoa had to complete their maturation either within the estrogen dominated female reproductive tract or in simple defined media containing a protein source, calcium and bicarbonate. In biological terms capacitation enables the spermatozoa to respond to physiological signals given out by the cumulus—oocyte complex by undergoing the acrosome reaction, an exocytotic event that releases enzymes involved in penetration of the egg investments. This process also results in the remodeling of the sperm surface such that it becomes capable of recognizing and fusing with the vitelline membrane of the oocyte (Yanagimachi, 1994). Thus, uncapacitated cells cannot respond to physiological agonists such as progesterone and ZP3 by undergoing the acrosome reaction and initiating oocyte fusion; this property is only expressed by capacitated cells.

Signal Transduction Mechanisms Regulating Sperm Acrosomal Exocytosis

Abstract: Publisher Summary This chapter reviews the signal transduction mechanisms regulating sperm acrosomal. The acrosome is a secretory vesicle overlying the nucleus of the mature spermatozoon. This organelle is a product of the Golgi complex, and is synthesized and assembled during spermiogenesis. The primary function of acrosomal exocytosis following zona pellucida adhesion is to permit the penetration of this unique extracellular matrix. This matrix induced exocytotic reaction is a regulated event that displays discrete properties. A consequence of zona pellucida penetration is the development of a sharply defined, thin penetration slit that is presumed to result from the limited digestion of the matrix by acrosome-associated proteases. If capacitation is necessary for spermatozoa to undergo physiologically relevant acrosomal exocytosis in response to the zona pellucid, it is reasonable that both the male and female reproductive tracts could influence capacitation. Factors comprising the reproductive tract fluids are thought to bind to and stabilize the sperm plasma membrane, presumably to prevent capacitation from occurring. Other factors remove molecules from the sperm surface, presumably to initiate the capacitation process. Currently, the understanding of the molecular mechanics of acrosomal exocytosis is at a very early stage. Some of the general mechanisms will be conserved with other exocytotic systems, but the sperm acrosome clearly has properties that distinguish it from other secretory systems, and it is these differences that will yield exciting results.

Utility of oxidative stress test in the male infertility clinic

Abstract: The controlled generation of very low amounts of reactive oxygen species (ROS) appears to regulate normal sperm functions, while high levels of ROS endanger sperm viability and function. Oxidative stress (OS) develops as a consequence of excessive production of ROS and/or impaired antioxidant defense system. It is proposed that such OS precipitates a range of pathologies currently thought to afflict male reproductive function. ROS-mediated peroxidative damage to the sperm plasma membrane may account for defective sperm function observed in a high proportion of infertility patients. Excessive generation of ROS may also attack integrity of DNA in the sperm nucleus. DNA bases are susceptible to oxidative stress, and peroxidation of these structures can cause base modification, DNA strand breaks and chromatin cross-linking. DNA damage induced by excessive ROS may accelerate the process of germ cell apoptosis, leading to decline in sperm counts associated with male infertility, and may explain the apparent deterioration of semen quality observed during the past four to five decades. For almost a decade, our research team in the Cleveland Clinic Foundation has identified the critical role of OS in male infertility. The main objective of our research was to transfer this important knowledge from the research bench to clinical practice. We designed studies with the aims of: 1. understanding the exact mechanisms by which OS develops in semen, which we thought will help setup strategies to overcome the problem, 2. establishing assays for accurate assessment of OS status and running the quality control studies for this purpose, 3. testing the correlation between OS and sperm nuclear DNA damage, and 4. identifying the clinical significance of seminal OS assessment in male infertility practice.

Distinct Membrane Fractions from Mouse Sperm Bind Different Zona Pellucida Glycoproteins

Abstract: Interactions between sperm and zona pellucida (ZP) during mammalian fertilization are not well characterized at the molecular level. To identify sperm proteins that recognize ligand ZP3, we used sonicated sperm membrane fractions as competitors in a quantitative binding assay. Sonicated membranes were density fractionated into 4 fractions. Bands 1‐3 contained membrane vesicles, and band 4 contained axonemal and midpiece fragments. In competitive binding assays, bands 1, 2, and 3 but not band 4 were able to compete with live, capacitated, intact sperm for soluble 125 I-ZP binding. Affinity-purified ZP fractions consisting of a ZP3-enriched fraction ( 125 I-ZP3) and a fraction enriched for ligands ZP1 and ZP2 and depleted of ZP3 ( 125 I-ZP1/ 2) were obtained by antibody affinity purification of ZP3. In competitive binding assays, bands 2 and 3 competed for 125 I-ZP3 binding, but band 1 did not interact with enriched 125 I-ZP3. None of the membrane fractions competed for 125 I-ZP1/2 binding. These results demonstrate that band 2 and band 3 contain sperm components that interact with ZP3 alone and that components in band 1 interact with ZP3 in conjunction with either ZP1 or ZP2. These data indicate that there must be at least 2 unique sperm plasma membrane components that mediate intact sperm interactions with ZP glycoproteins in mouse. Bands 2 and 3 are likely to contain a primary ZP-binding protein because they interacted directly with ZP3, whereas band 1 may contain sperm proteins involved in later interactions with the ZP, perhaps transitional interactions to maintain sperm contact with the ZP during acrosomal exocytosis. acrosome reaction, fertilization, gamete biology, signal transduction, sperm

Binding of protein D/E to the surface of rat epididymal sperm before ejaculation and after deposition in the female reproductive tract.

Abstract: The objectives of the present investigation were to study the interaction of protein D/E with the surface of rat epididymal spermatozoa and to assess its topology on the spermatozoa surface before and after deposition in the female reproductive tract. Protein D/E, a member of the cysteine-rich secretory protein (CRISP-1) family, has been proposed to be involved in sperm-egg membrane fusion. In vitro competitive photoactivated cross-linking experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis revealed that protein D/E molecules specifically interact with two surface proteins exhibiting an M(r) approximately 120.0 kd and approximately 130.0 kd, respectively, on the sperm surface. In vitro treatment of epididymal spermatozoa with phosphatidylinositol specific-phospholipase C revealed the release of protein D/E molecules over the head region but not the tail region of spermatozoa. Indirect immunofluorescence experiments using polyclonal antibodies generated against a highly purified protein D/E preparation demonstrated that protein D/E molecules were bound to the surface of spermatozoa recovered from the epididymal and female reproductive tracts, even after 7 hours. These results indicate that protein D/E molecules interact with specific membrane proteins, and is subsequently covalently bound to the surface of spermatozoa via a glycosyl-phosphatidyl inositol linkage. In addition, protein D/E molecules remain covalently bound to spermatozoa after deposition in the female reproductive tract, an observation that is consistent with the proposed physiological function of the protein in the fertilization process.

Rabbit epididymal secretory proteins. II. Immunolocalization and sperm association of REP38

Abstract: Polyclonal antibody was used to partially characterize REP38, a major rabbit epididymal secretory protein. Western blot analyses and immunohistochemistry indicated that REP38 is only expressed in regions 5 and 6 of the epididymis (corpus epididy-midis) and is localized in the supranuclear region and microvilli of the principal cells in these regions. It was not expressed in other tissues of the body. In region 8 (cauda epididymidis), REP38 was detected in the luminal border and cytoplasm of scattered principal cells, indicating that it may be reabsorbed in this region. This protein accumulated on the sperm plasma membrane downstream of region 5 and was localized predominantly over the acrosomal and postacrosomal regions of the head and the middle piece. Although tightly bound to epididymal sperm, REP38 migrated to the equatorial segment under conditions in vivo that would promote capacitation. When tested in vitro, anti-REP38 IgG reduced the percentage of ova fertilized in a concentration-dependent manner, apparently by blocking sperm-egg fusion.

N-acetyl beta-D-glucosaminidase is not attached to human sperm membranes through the glycosylphosphatidyl inositol (GPI)-anchor.

Abstract: Aim: The mode of anchorage of N-acetyl β-D-glucosaminidase (NAGA) on human ejaculated sperm was investigated. Methods: Sperm plasma membrane was prepared by discontinuous sucrose gradient centrifugation from human sperm. NAGA was solubilized from these membranes by two detergents: octyl-glycoside and triton X-100. In separate studies, the release of the enzyme from the sperm membrane preparation by phosphatidylinositol specific phospholipase C (PI-PLC) was also examined. Results: NAGA activity was detected on sperm membranes isolated from human ejaculates. The pattern of the enzyme solubilization by detergents indicated that the enzyme was an integral protein of sperm membrane. NAGA was not released from the sperm membranes by PI-PLC treatment. Conclusion: The evidence presented strongly suggests that human sperm membrane bound NAGA is not attached via the GPI anchor.